Methods for characterizing lentiviruses

Abstract

The present disclosure provides a method of characterizing a lentivirus, the method comprising: providing a sample comprising a lentiviral population, the lentiviral population comprising a fully loaded lentivirus, a partially loaded lentivirus, and / or an empty lentivirus; contacting a substrate with the sample to capture the lentiviral population; contacting the captured lentiviral population with a first fluorescent agent comprising a first fluorescent label and a first binding molecule that binds to an envelope protein of the lentivirus, a second fluorescent agent comprising a second fluorescent label and a second binding molecule that binds to a capsid protein of the lentivirus, and a third fluorescent agent, the third fluorescent agent comprises a third fluorescent label bound to the payload; irradiating the captured lentiviral population with light to excite the fluorescent agent; detecting fluorescent light rays of different wavelengths emitted from the fluorescent agent; and characterizing the lentivirus according to the detected fluorescent light.

Description

Technical Field

[0001] The present disclosure provides methods for characterizing lentiviral vectors using multiple fluorophores that bind to the envelope protein, capsid protein, and / or payload of the lentiviral vector, respectively. By detecting fluorescence at different wavelengths, fully loaded lentiviruses, partially loaded lentiviruses, and empty lentiviruses can be identified and quantified. The method provides rapid, simple, and simultaneous characterization of lentiviral vectors, which aids in the optimization of lentiviral vector production. Background Art

[0002] Lentiviral vectors are biological carriers that can be used to deliver therapeutic genes ("payloads") into cells. Current lentiviral manufacturing processes typically produce only about 20% fully loaded lentiviral vectors that contain the three viral components and the payload. A large content of empty or partially loaded lentiviral vectors reduces the efficacy of lentivirus-mediated gene delivery while increasing the immunogenic burden on transduced cells in vitro or in vivo, which may trigger serious adverse events in patients. There is a need for a method to identify fully loaded lentiviral vectors as well as partially loaded vectors. The present invention meets this need. Summary of the Invention

[0003] In some embodiments, provided herein is a method for characterizing a lentiviral vector, the method comprising: providing a sample comprising a population of lentiviral vectors, the population of lentiviral vectors comprising fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors; contacting a substrate with the sample to thereby capture the population of lentiviral vectors on the substrate, wherein the substrate comprises an anti-envelope protein antibody; contacting the captured population of lentiviral vectors with a first fluorophore, a second fluorophore, and a third fluorophore, the first fluorophore comprising a first fluorescent label and a first binding molecule that binds to the envelope protein of the lentiviral vector, the second fluorophore comprising a second fluorescent label and a second binding molecule that binds to the capsid protein of the lentiviral vector, the third fluorophore comprising a third fluorescent label that binds to the payload of the lentiviral vector; irradiating the captured population of lentiviral vectors with light from an irradiation source to thereby excite the first fluorophore, the second fluorophore, and the third fluorophore; detecting a first fluorescent light of a first fluorescent wavelength emitted from the first fluorophore, a second fluorescent light of a second fluorescent wavelength emitted from the second fluorophore, and a third fluorescent light of a third fluorescent wavelength emitted from the third fluorophore; and characterizing the lentiviral vector based on the detected first fluorescent light, second fluorescent light, and third fluorescent light, wherein the first fluorescent wavelength, the second fluorescent wavelength, and the third fluorescent wavelength are different. Brief Description of the Drawings

[0004] Figure 1Shows the structure of a lentiviral vector with a payload.

[0005] Figure 2 Shows a three - layer visualization method of a lentiviral vector according to some embodiments of the present disclosure.

[0006] Figure 3 Shows packaging plasmids, envelope plasmids, and transfer plasmids for generating lentiviral vectors according to some embodiments of the present disclosure.

[0007] Figure 4 Shows different types of capsid loading of recombinant adeno - associated virus (rAAV) in rAAV production.

[0008] Figure 5 Shows a lentiviral vector map generated using three different ratios of plasmids according to some embodiments of the present disclosure.

[0009] Figure 6 Shows a lentiviral vector map generated using five different ratios of plasmids according to some embodiments of the present disclosure, which expresses green fluorescent protein (GFP). Detailed Description

[0010] In the claims and / or the specification, when the term "comprising" is used, the use of the word "a / an" can mean "one", but is also consistent with "one or more", "at least one", and "one or more than one".

[0011] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variations of the method / device for measuring the value. Generally, depending on the circumstances, the term is intended to cover a variability of about or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0012] The use of the term "or" in the claims is used to mean "and / or", unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although the present disclosure supports definitions that refer only to alternatives as well as to "and / or".

[0013] As used in this specification and the claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

[0014] As used herein, "nucleic acid", "nucleic acid molecule" or "oligonucleotide" means a polymeric compound comprising covalently linked nucleotides. The term "nucleic acid" includes polyribonucleic acid (RNA) and polydeoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA. RNA includes, but is not limited to, mRNA, tRNA, rRNA, snRNA, microRNA, miRNA or MIRNA.

[0015] As used herein, "gene" refers to an assembly of nucleotides encoding a polypeptide and includes cDNA and genomic DNA nucleic acid molecules. "Gene" also refers to nucleic acid fragments that can serve as regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence) the coding sequence. In some embodiments, the gene is integrated with multiple copies. In some embodiments, the gene is integrated at a predefined copy number.

[0016] As Figure 1 As shown, a functional lentiviral vector (also referred to herein as a functional lentivirus) has three key components: an outer envelope, which is composed of, for example, vesicular stomatitis virus glycoprotein (VSV-G); a capsid, which houses the genetic material and is composed of, for example, p24 protein; and a payload and / or genomic RNA, typically a therapeutic nucleic acid. There is currently no single assay for comprehensively understanding the state of lentiviral vectors after production. Current assays are time-consuming, low-throughput, and generally labor-intensive. In addition, most assays only measure a single property of the lentiviral vector, rather than the complete vector structure.

[0017] In certain aspects, the present disclosure provides a method for characterizing lentiviral vectors using three-layer visualization. In some embodiments, the method does not require purification of the lentiviral vector from the culture medium and can be performed at high throughput to analyze multiple samples simultaneously.

[0018] In some embodiments, a method of characterizing a lentiviral vector includes: providing a sample comprising a population of lentiviral vectors, the population of lentiviral vectors comprising fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors; contacting a substrate with the sample to capture the population of lentiviral vectors on the substrate, wherein the substrate comprises an anti-envelope protein antibody; contacting the captured population of lentiviral vectors with a first fluorophore, a second fluorophore, and a third fluorophore, the first fluorophore comprising a first fluorescent label and a first binding molecule that binds to the envelope protein of the lentiviral vector, the second fluorophore comprising a second fluorescent label and a second binding molecule that binds to the capsid protein of the lentiviral vector, and the third fluorophore comprising a third fluorescent label that binds to the payload. The method further includes irradiating the captured population of lentiviruses with light from an irradiation source to excite the first fluorophore, the second fluorophore, and the third fluorophore. The method further includes detecting a first fluorescent light of a first wavelength emitted from the first fluorophore, a second fluorescent light of a second wavelength emitted from the second fluorophore, and a third fluorescent light of a third fluorescent wavelength emitted from the third fluorophore; and then characterizing the lentivirus based on the detected first fluorescent light, second fluorescent light, and third fluorescent light. In some embodiments, the first, second, and third fluorescent wavelengths are different.

[0019] Figure 2 A three-layer visualization method of a lentiviral vector according to some embodiments of the present disclosure is shown, wherein a fluorescent anti-VSV-G antibody, a fluorescent anti-p24 antibody, and a fluorescent nucleic acid stain are used to provide three-layer visualization. In some embodiments, the present disclosure targets the payload by using a permeable fluorescent nucleic acid stain that selectively stains the RNA payload.

[0020] Suitably, the lentiviral sample is contacted with a substrate (such as a microarray chip comprising an envelope capture antibody (such as an anti-VSV-G capture antibody)). As Figure 2 shown, the capture antibody captures the lentiviral vector via the interaction between the surface-bound antibody and the antibody on the outside of the lentiviral vector. Exemplary envelope capture antibodies include, for example, VSV-G capture antibodies, F, and HN antibodies, as described herein. The captured and bound sample is fixed, permeabilized, and stained with a mixture containing fluorescent antibodies (which target the envelope and capsid, such as fluorescent anti-VSV-G and fluorescent anti-p24) and a permeable fluorescent nucleic acid stain that targets the payload. The selected fluorescent nucleic acid stain is selective for nucleic acids (which have a high affinity for RNA) and is non-fluorescent when not bound to nucleic acids. The processed sample is then scanned on a fluorescence imaging platform, where the co-localization of all three fluorescent signals is monitored. The co-localization of the three fluorescent signals indicates the percentage of complete lentiviruses in the sample. By this method, the present disclosure can visualize the characteristics of the lentiviral vector at three separate levels to gain a comprehensive understanding of the particle. In some embodiments, the fluorescence imaging platform is Imaging platform.

[0021] The methods described herein utilize fluorescence visualization of lentiviral characteristics at three different levels (envelope, capsid, and payload) and indicate the colocalization of these three attributes in a sample at the single virion level. The results provide the percentage of truly complete and potentially functional particles in a small volume and high-throughput manner. This disclosure is advantageous because there are currently no available methods on the market that can provide all the information in one assay. Currently available assays only measure a single attribute of the lentivirus, and data needs to be pieced together to gain a comprehensive understanding of the particle.

[0022] The terms "lentivirus," "lentiviral vector," and "lentiviral particle" are used interchangeably herein. As used herein, a "lentiviral vector" refers to a vector that can be inserted into a desired gene and is suitable for use in research or therapeutic applications for gene therapy purposes. Suitably, the lentiviral vector is from the HIV family. Lentiviral vectors are a well-studied vector system based on the human immunodeficiency virus (HIV-1). Since lentiviral vectors integrate into the host cell genome, this vector allows for persistent transgene expression. Other lentiviral systems have also been developed as gene transfer systems, including HIV-2 simian immunodeficiency virus, non-primate lentiviruses, feline immunodeficiency virus, and bovine immunodeficiency virus, among others. Due to the pathogenicity of HIV-1 in humans, for safety reasons, the most widely used lentiviral system for clinical and research and development purposes is based on a four-plasmid system (third-generation lentiviral vector), which expresses the lentiviral group-specific antigen (GAG) and lentiviral polymerase (POL) proteins, respectively; an envelope protein (usually vesicular stomatitis virus glycoprotein (VSV-G)); the REV protein (a protein that regulates the expression of HIV viral particles); and a transfer vector (TV) containing the gene of interest (GOI). The GOI can be introduced into a desired cell for therapy and disease treatment, including immunodeficiency and neurodegenerative diseases. In some embodiments, other types of lentiviral vectors can also be used, such as the second-generation system with three vectors.

[0023] In some embodiments, lentiviral vectors are produced by transfecting host cells using the plasmids described above. As used herein, "transfection" means introducing an exogenous nucleic acid molecule, including a plasmid and / or vector, into a cell. A "transfected" cell includes the exogenous nucleic acid molecule inside the cell, and a "transformed" cell is a cell in which the exogenous nucleic acid molecule inside the cell induces a phenotypic change in the cell. The transfected nucleic acid molecule can integrate into the genomic DNA of the host cell and / or can be maintained by the cell extrachromosomally, either transiently or for a long time. In some embodiments, "transduction" means infecting mammalian cells with a viral vector and is used interchangeably with "transfection" in this disclosure.

[0024] A host cell or organism that expresses an exogenous nucleic acid molecule or fragment is referred to as a "recombinant", "transformed", or "transgenic" organism. Suitably, host cells that can be used in the various methods described herein are mammalian cells and cell lines or cultures. As used herein, the term "mammalian cell" encompasses cells from any member of the mammalian order, such as human cells, mouse cells, rat cells, monkey cells, hamster cells, etc. In some embodiments, the cells are mouse cells, human cells, Chinese hamster ovary (CHO) cells, CHOK1 cells, CHO-DXB11 cells, CHO-DG44 cells, CHOK1SV cells (including all variants (e.g., Lonza, Slough, UK), CHOK1SV GS-KO (glutamine synthetase knockout) cells (including all variants (e.g., XCEED TM Lonza, Slough, UK)). Exemplary human cells include human embryonic kidney (HEK) cells, such as HEK-293, HeLa cells, or HT1080 cells. In some embodiments, the lentiviral vectors of the present disclosure are produced from HEK-293 cells, human Caucasian colon adenocarcinoma HT-29 cells, or mesenchymal stem cells (MSCs).

[0025] Mammalian cells include mammalian cell cultures, which can be adherent cultures or suspension cultures. An adherent culture refers to cells that grow on a substrate surface (e.g., a plastic plate, culture dish, or other suitable cell culture growth platform) and can be anchorage-dependent. A suspension culture refers to cells that can be maintained in, for example, a culture flask or large suspension bucket, which allows for a larger surface area for gas and nutrient exchange. Suspension cell cultures typically utilize a stirring or agitation mechanism to provide adequate mixing. The media and conditions for maintaining cells in suspension are generally well known in the art. Exemplary suspension cell cultures include human embryonic kidney (HEK293) clonal cells.

[0026] In some embodiments, the lentiviral vectors of cells and their products are produced in a bioreactor. The cells can be prepared in any suitable bioreactor (also referred to herein as a reactor), including but not limited to stirred tank, air-lift, fibrous, microfibrous, hollow fiber, ceramic matrix, fluidized bed, fixed bed, and / or spouted bed bioreactors. As used herein, "bioreactor" can include a fermenter or fermentation unit or any other reaction vessel, and the terms "bioreactor" and "reactor" are used interchangeably with "fermenter". The term fermenter or fermentation refers to both microbial cultures and mammalian cultures. For example, in some aspects, an exemplary bioreactor unit can perform one or more or all of the following: feeding of nutrients and / or carbon sources, injection of suitable gases (e.g., oxygen), inlet and outlet flow of fermentation or cell culture medium, separation of gas and liquid phases, maintenance of temperature, maintenance of oxygen and CO2 levels, maintenance of pH level, agitation (e.g., stirring), and / or cleaning / sterilization. An exemplary reactor unit, such as a fermentation unit, can contain multiple reactors within the unit. For example, the unit can have 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors in each unit, and / or a facility can contain multiple units having single or multiple reactors within the facility. In various embodiments, the bioreactor can be suitable for batch, semi-batch, fed-batch, perfusion, and / or continuous fermentation processes. Any suitable reactor diameter can be used. In an embodiment, the volume of the bioreactor can be between about 100 mL and about 50,000 L. Non-limiting examples of volumes are 100 mL, 250 mL, 500 mL, 750 mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and / or 50,000 liters.Alternatively, suitable reactors can be reusable, single-use, disposable, or non-disposable, and can be formed from any suitable material, including metal alloys such as stainless steel (e.g., 316L or any other suitable stainless steel) and Inconel, plastics, and / or glass.

[0027] In some embodiments, the host cell produces a lentiviral vector population. In some embodiments, as Figure 3 shown, the host cell is transduced using two packaging plasmids, an envelope plasmid, and a transfer plasmid. Depending on the type and ratio of the plasmids, the host cell, the culture conditions of the host cell, the ion exchange polishing for lentiviral vector enrichment, and the size exclusion chromatography for removing damaged lentiviral particles, the resulting lentiviral vector population can include different ratios of fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors.

[0028] As used herein, a fully loaded lentiviral vector refers to a vector that includes envelope proteins formed into a lentiviral envelope, capsid proteins formed into a lentiviral capsid, and also includes one or more copies of a payload sufficient for payload delivery. As used herein, a partially loaded lentiviral vector refers to a vector that includes envelope proteins (formed into an envelope or a partial envelope) and capsid proteins (formed into a capsid or a partial capsid), but only has a fragment of the payload or host cell DNA or RNA. The host cell DNA / RNA or partial payload may not be suitable for research or therapeutic applications. As used herein, an empty lentiviral vector refers to a vector that includes envelope proteins (formed into an envelope or a partial envelope) and capsid proteins (formed into a capsid or a partial capsid), but is substantially lacking in payload. In some embodiments, the lentiviral vector population can further include other components or impurities, such as small aggregates formed from envelope proteins or capsid proteins. These aggregates do not contain payload.

[0029] Figure 4An overview of the main types of capsids generated during the production of recombinant adeno-associated virus (rAAV), where rAAV may not include an envelope, while fully loaded lentiviral vectors have an envelope (Gimpel et al., Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies, Molecular Therapy Methods & Clinical Development, 2021, 20:740-754). It serves as an example of different scenarios for viral genome packaging, as the process has been well studied in AAV but is less understood in lentiviruses. As Figure 4 shown, full capsids have correct genome packaging, partially filled capsids only package part of the vector or host cell DNA, and empty capsids do not have a packaged genome. Although the percentage of full capsids during harvest is less than 30%, it can be increased to over 70% after purification. As described herein, and as Figure 4 illustrated in, partially filled capsids, empty capsids, and aggregates not only fail to provide sufficient transduction of the desired payload, but injecting them into a patient may increase the risk of immunotoxicity.

[0030] As discussed above, a sample (such as a cell culture with a lentiviral vector) can be contacted with a substrate such that the substrate captures the lentiviral vector from the sample. The substrate (see Figure 2 ) is preferably an optical substrate configured to generate an enhanced fluorescence signal to improve the sensitivity of lentiviral vector detection. In some embodiments, the substrate has a flat reflective surface and an anti-envelope protein antibody is immobilized on the surface. In some embodiments, the flat surface of the substrate may include one or more layers that enhance the excitation and / or emission of fluorescent light from a first fluorescent label, a second fluorescent label, and a third fluorescent label. In some embodiments, the substrate is a silicon substrate, such as a glass slide. In some embodiments, the substrate comprises an oxide layer on silicon. In some embodiments, the substrate is a microarray chip. In some embodiments, the substrate is a bead, such as a magnetic bead. In some embodiments, each chip is suitable for characterizing lentiviral vectors in one sample; and multiple chips can be placed in a multi-well plate (such as a 6-well, 24-well, 48-well, or 96-well plate) such that multiple samples corresponding to multiple substrates or chips can be characterized simultaneously, and preferably automatically. In some embodiments, each chip may have multiple spots for characterizing multiple samples.

[0031] In some embodiments, the reflectivity of the substrate at one or more wavelengths and / or spectral ranges of interest is greater than a specific minimum value. Exemplary spectral ranges include, but are not limited to, the UV spectral range (ranging from about 400 nm to 450 nm), the blue spectral range (ranging from about 460 nm to 500 nm), the green spectral range (ranging from about 520 nm to 560 nm), the red spectral range (ranging from about 640 nm to 680 nm), and the deep red spectral range (ranging from about 710 nm to 750 nm). For example, the "reflectivity" or "reflectance" of the reflective substrate at one or more wavelengths and / or spectral bands of interest can be greater than or approximately equal to 25% (e.g., greater than 30%, e.g., greater than 40%, e.g., greater than 50%, e.g., greater than 60%, greater than 70%). In certain embodiments, the reflectivity of the reflective substrate at one or more wavelengths and / or spectral bands of interest is greater than 80% or higher. In some embodiments, the reflective substrate comprises an oxide layer on a silicon base. In certain embodiments, the reflective substrate comprises multiple layers.

[0032] As discussed above, the substrate surface is coated with an anti-envelope protein antibody such that it is immobilized on the substrate surface. The envelope protein of the lentiviral vector can be at least one of VSV-G, a fusion protein, hemagglutinin, and hemagglutinin-neuraminidase (HN). Correspondingly, the substrate is immobilized with at least one of an anti-VSV-G antibody, an anti-fusion protein (anti-F) antibody, an anti-hemagglutinin (anti-HA) antibody, and an anti-HN antibody. Thus, a substrate with an anti-envelope antibody can capture a lentiviral vector with a corresponding envelope protein. In some embodiments, the lentiviral vector has a VSV-G envelope protein, and the substrate surface is coated with an anti-VSV-G antibody. In some embodiments, the anti-VSV-G antibody is attached to the substrate surface by physical adsorption, chemical linkage, or biological means.

[0033] In some embodiments, the contact between the sample and the substrate surface is achieved by incubating the sample and the substrate together at a desired temperature for a predetermined period of time. In some embodiments, the incubation is carried out at 20 °C, 25 °C, 30 °C, 35 °C or 37 °C. In some embodiments, the predetermined period of time is from about 5 minutes to about 90 minutes. In some embodiments, the predetermined period of time is from about 15 minutes to about 60 minutes. In some embodiments, the predetermined period of time is about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes or about 90 minutes. In some embodiments, the predetermined time is about 15 minutes. In some embodiments, the predetermined time is about 30 minutes. In some embodiments, the predetermined time is about 60 minutes. After incubation, the lentiviral population from the sample is immobilized on the substrate surface via the binding of the VSV-G protein of the lentiviral vector to the anti-VSV-G antibody on the substrate surface. In some embodiments, after incubation, the contacted sample (and thus unbound viral vectors or segments / aggregates) can be removed, for example, by simply lifting the substrate to decant the sample solution and then optionally drying the substrate for a short period of time, such as about 5 minutes to about 10 minutes.

[0034] After the sample contacts the substrate, the method further contacts the substrate with a first fluorophore, a second fluorophore, and a third fluorophore. Optionally, the method can contact the sample and the three fluorophores with the substrate simultaneously, or contact the sample and two of the three fluorophores with the substrate simultaneously. The fluorophores comprise fluorescent labels.

[0035] As used herein, the term "label" or "tag" refers to a composition capable of generating a detectable signal indicative of the presence of a target in an assay sample. Suitable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties, etc. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. In some embodiments, the label is a fluorescent label comprising a fluorescent molecule.

[0036] In some embodiments, the first fluorophore comprises a first fluorescent label and a first binding molecule. The first binding molecule is configured to target the lentiviral envelope protein of the lentiviral vector. Suitably, the first binding molecule can be an anti-VSV-G antibody, an anti-F antibody, an anti-HA antibody or an anti-HN antibody. In some embodiments, the first binding molecule is an anti-VSV-G antibody. In some embodiments, the first binding molecule is the same as the capture molecule on the substrate, except that the capture molecule is immobilized on the substrate surface while the first binding molecule is linked to the first fluorescent label.

[0037] In some embodiments, the second fluorophore comprises a second fluorescent label and a second binding molecule. The second binding molecule is configured to target the capsid protein of the lentiviral vector. Suitably, the second binding molecule can be an anti-p7 antibody, an anti-p24 antibody, or an anti-p27 antibody. In some embodiments, the second binding molecule is an anti-p24 antibody.

[0038] In some embodiments, the third fluorophore comprises a third fluorescent label. The third fluorescent label is configured to bind to the payload of the lentiviral vector. The payload can be a nucleic acid (such as DNA or RNA) or a protein. Correspondingly, the third fluorescent label can be a DNA stain, an RNA stain, a nucleic acid stain, or an antibody targeting the payload protein. In some embodiments, the third fluorescent label can also be an anti-integrase antibody, an anti-reverse transcriptase antibody, or an anti-protease antibody targeting these different functional proteins.

[0039] In some embodiments, the sample is diluted using a suitable buffer before contacting the substrate. In some embodiments, the sample is diluted to at least about 1:10, 1:20, 1:50, 1:100, 1:200, 1:500, 1:1000; 1:1500, 1:2000. In some embodiments, the dilution is such that the concentration of the lentiviral vector (lentiviral particles) in the sample is about 10 5 to 10 8 per milliliter (ml). In some embodiments, the concentration of the lentiviral vector is about 10 6 to 10 8 / ml. In certain embodiments, the concentration of the lentiviral vector in the sample is estimated by any suitable means (such as interference microscopy, flow cytometry, fluorescence spectroscopy, titer determination, polymerase chain reaction (PCR)), or estimated based on previous experiments and experience.

[0040] In some embodiments, after the substrate contacts the sample, the first fluorophore, the second fluorophore, and the third fluorophore then contact the substrate simultaneously. In some embodiments, after the substrate contacts the sample, the substrate first contacts the third fluorophore and then contacts the second and third fluorophores. In some embodiments, the third fluorophore comprises a permeable fluorescent nucleic acid stain to permeabilize the stain into the lentiviral vector. In some embodiments, the third fluorophore comprises a fluorescent nucleic acid stain and a permeabilizing reagent, and the lentiviral vector immobilized on the substrate is treated with the permeabilizing reagent for a period of time and then contacts the fluorescent nucleic acid stain. Alternatively, the permeabilizing reagent and the fluorescent nucleic acid stain contact the lentiviral vector simultaneously for a period of time. The permeabilizing reagent can be, for example, an organic solvent (such as methanol or acetone), a detergent (such as Triton-X, Tween, or NP-40), a selective detergent (such as saponin, digitonin, or leucopem). In some embodiments, the permeabilization period is less than 5 hours, less than 4 hours, less than 3 hours, less than 2 hours, or less than 1 hour. In some embodiments, the permeabilization period is about 0.5 hour to 2 hours. In some embodiments, the permeabilization period is less than 30 minutes, less than 15 minutes, or less than 10 minutes. By limiting the permeabilization period, damage to the lentiviral vector is prevented or significantly reduced.

[0041] In some embodiments, the step of contacting the captured lentiviral vector population is carried out by incubating the substrate having the captured lentiviral vector population with the first fluorophore, the second fluorophore, and the third fluorophore for a period of time (incubation period). In some embodiments, the incubation time is about 15 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. In some embodiments, the incubation time is about 15 - 60 minutes. In some embodiments, the incubation period is at least 15 minutes. In some embodiments, the incubation is about 30 minutes. In some embodiments, the incubation is carried out at a temperature of about 15°C - 40°C. For example, at a temperature of about 15°C - 40°C, such as at about 20°C, about 25°C, or about 37°C, the substrate can be contacted with the fluorophore for about 30 minutes to about 2 hours, or about 30 minutes to about 1.5 hours, or about 45 minutes to about 1.5 hours, or about 1 hour to about 1.5 hours, or about 1.5 hours.

[0042] After labeling with the three fluorophores, the substrate having the captured lentiviral population can be washed one or more times to remove unbound fluorophores and other debris. In some embodiments, no washing step is required.

[0043] After the substrate contacts the sample and the first, second, and third fluorophores, the lentiviral population captured on the substrate surface is linked to the fluorescent label and can be detected using fluorescence.

[0044] In some embodiments, a fluorescence detection device is used for fluorescence characterization of lentiviral vectors. The fluorescence detection device suitably includes an illumination light source, a fluorescence detector, a computing device, and optionally an interference microscope.

[0045] The illumination light source generates excitation light, and the excitation light is directed onto the substrate surface such that the fluorescent labels can emit fluorescence signals after being activated by the excitation light. In some embodiments, the illumination light source includes one or more light-emitting diodes (LEDs) or one or more lasers. In some embodiments, the light source is a coherent light source that generates light of a specific wavelength. In some embodiments, the illumination light source generates three specific wavelengths of light corresponding to three fluorescent labels. In some embodiments, each specific wavelength is a narrow range of wavelengths. In some embodiments, the wavelength of the excitation light can be, for example, from about 400 nm to about 450 nm (UV), from about 460 nm to about 500 nm (blue), from about 520 nm to about 560 nm (green), from about 640 nm to about 680 nm (red), or from about 710 nm to about 750 nm (deep red). In some embodiments, the wavelength of the illumination light source is adjustable. In some embodiments, the illumination light source has three to five channels, each channel being configured to illuminate a coherent light source of a predetermined wavelength.

[0046] After absorbing the excitation light, the first fluorescent label, the second fluorescent label, and the third fluorescent label in the lentiviral vector are configured to emit light of a first fluorescence wavelength, a second fluorescence wavelength, and a third fluorescence wavelength, respectively. In some embodiments, each of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range of about 250 nm to about 700 nm. In some embodiments, one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range of about 460 nm to about 510 nm; one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range of about 520 nm to about 570 nm; and one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range of about 640 nm to about 680 nm.

[0047] In some embodiments, the emission wavelengths of the first fluorescent label, the second fluorescent label, and the third fluorescent label are in the UV range of about 330 nm to about 380 nm, in the blue light range of about 420 nm to about 495 nm, in the yellow light range of about 520 nm to about 580 nm, or in the red light range of about 620 nm to about 750 nm. In some embodiments, the difference in emission wavelengths between two of the first fluorescent label, the second fluorescent label, and the third fluorescent label (i.e., the difference between the emission wavelengths) is at least about 20 nm to about 80 nm. In some embodiments, the difference is at least about 40 nm to 60 nm. In some embodiments, the difference is about 50 nm. In some embodiments, the emission wavelengths of the first fluorescent label, the second fluorescent label, and the third fluorescent label are about 460 nm to about 510 nm, about 520 nm to about 570 nm, and about 640 nm to about 680 nm.

[0048] In some embodiments, the wavelength of the excitation light of the first of the first fluorescent label, the second fluorescent label, and the third fluorescent label is about 488 nm, and the corresponding emission wavelength is about 510 nm. In some embodiments, the first fluorescent label is the NovaFluor Blue 510 dye from THERMOFISHER. In some embodiments, the wavelength of the excitation light of the second fluorescent label is about 561 nm, and the corresponding emission wavelength is about 568 nm. In some embodiments, the second fluorescent label is the NovaFluor Yellow 570 dye from THERMOFISHER. In some embodiments, the wavelength of the excitation light of the third fluorescent label is about 640 nm, and the corresponding emission wavelength is about 685 nm. In some embodiments, other types of fluorescent labels or fluorescent label / excitation light combinations can be used, as long as the emission lights of the three fluorescent labels are easily distinguishable. In some embodiments, suitable dyes are 555 (VSV-G) and 647 (p24).

[0049] In some embodiments, the first fluorescent label is attached chemically or biologically to the first binding molecule, and the second fluorescent label is attached chemically or biologically to the second binding molecule.

[0050] In some embodiments, the third fluorescent label is a nucleic acid stain. The nucleic acid dye can be, for example, Quant-iT TM PicoGreen dsDNA stain, Quant-iT TM OliGreen ssDNA stain, Quant-iT TM RiboGreen RNA stain, Green fluorescent nucleic acid stain, green fluorescent nucleic acid stain, Hoechst 33258 blue fluorescent nucleic acid stain, SYBR TM Green I nucleic acid gel stain or SYTO TM RNASelect TM green fluorescent stain. In some embodiments, the third fluorescent label is a permeable fluorescent nucleic acid stain that penetrates the envelope and capsid of the lentiviral vector and selectively stains the RNA payload inside the capsid. In some embodiments, the third fluorescent label can also be carboxyfluorescein succinimidyl ester (6-carboxyfluorescein succinimidyl ester; 5(6)-CFDA-SE) (CFSE), a dye that couples via its succinimidyl group to lentiviral vector molecules intracellularly, specifically to intracellular lysine residues and other amine sources.

[0051] In some embodiments, interference microscopy is used to characterize the position and size of lentiviral vectors on a substrate surface. For example, an IM microscope scans the substrate surface to obtain an IM image, and a computing device uses the IM image to identify lentiviral vectors in the IM image. In some embodiments, particles smaller than 10 nm (diameter) are discarded; particles sized from about 10 nm to about 50 nm are labeled as fragments; and particles sized from about 50 nm to about 200 nm are labeled as lentiviral vectors. In some embodiments, fluorescence-based subsequent analysis is performed only on lentiviral particles larger than 50 nm.

[0052] The fluorescent light emitted from the first, second, and third fluorescent labels can be detected by a fluorescence detector or a spectrophotometer. In some embodiments, the detector is a charge-coupled device (CCD) camera. The CCD collects images of each sample at each wavelength; and for each sample, three corresponding images are collected substantially simultaneously at the same light-receiving location. Thus, the fluorescence signals at the corresponding locations cover the same sample material.

[0053] As defined herein, a "spectrophotometer" is a photometer (a device for measuring light intensity) that can measure light intensity as a function of color (or more specifically, wavelength). In some embodiments, the spectrophotometer is configured to measure the light intensity at the first emission wavelength, the second emission wavelength, and the third emission wavelength of the first, second, and third fluorescent labels. In some embodiments, the spectrophotometer is configured to measure the ratio of light intensities between the first emission wavelength, the second emission wavelength, and the third emission wavelength. One, two, or three spectrophotometers can be used to measure the three fluorescence light intensities or the ratio between the three fluorescence light intensities. In some embodiments, the present disclosure is implemented on the LENTIVIEW from NanoView BiosciencesTM Performed on an imaging platform. In some embodiments, the present disclosure is performed on the VIRUS from SARTORIUS Performed on an imaging platform, and the spectrophotometer can be, for example, Virus Counter 3100. In certain embodiments, the present disclosure can also be performed on the EXOVIEW TM Imaging platform suitable for lentiviral vector analysis.

[0054] In some embodiments, the spectrophotometer is configured to scan the fluorescence intensity image of the same area of the substrate surface at a first emission wavelength, a second emission wavelength, and a third emission wavelength (fluorescence wavelength) to obtain a first fluorescence image reflecting the fluorescence signal at the first emission wavelength, a second fluorescence image reflecting the fluorescence signal at the second emission wavelength, and a third fluorescence image reflecting the fluorescence signal at the third emission wavelength. Each fluorescence intensity image has a plurality of fluorescence signals, and each fluorescence signal in the fluorescence image can correspond to a lentiviral vector.

[0055] In some embodiments, the fluorescence device does not include an interference microscope, and the lentiviral vector is directly identified from the first fluorescence image. In some embodiments, the lentiviral vector is identified by an image target detection algorithm. For example, if the fluorescence signal of each pixel in the first fluorescence image is greater than a threshold, it is evaluated as a fluorescent pixel. Adjacent fluorescent pixels are combined to form a fluorescent spot, and if the longest diameter of the spot is greater than 50 nm, the spot represents a lentiviral vector. In some embodiments, the lentiviral vector can also be detected from each of the first fluorescence image, the second fluorescence image, and the third fluorescence image by an image target detection algorithm.

[0056] When the IM image is available, in some embodiments, three fluorescence images of each substrate surface are overlapped with the corresponding IM image to characterize the lentiviral vector. In some embodiments, for positions in the IM image that have lentiviral vectors, the computing device determines whether there are fluorescence signals at the first fluorescence image, the second fluorescence image, and the third fluorescence image. For each lentiviral vector or particle identified in the IM image, the computing device calculates a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal from the first fluorescence image, the second fluorescence image, and the third fluorescence image corresponding to the identified lentiviral vector, and compares the first fluorescence signal, the second fluorescence signal, and the third fluorescence signal with a first fluorescence threshold, a second fluorescence threshold, and a third fluorescence threshold. When the first fluorescence signal (intensity) is greater than the first fluorescence threshold, the identified lentiviral vector is considered to have envelope fluorescence. When the second fluorescence signal (intensity) is greater than the second fluorescence threshold, the identified lentiviral vector is considered to have capsid fluorescence. When the third fluorescence signal (intensity) is greater than the third fluorescence threshold, the identified lentiviral vector is considered to have payload fluorescence; and when the third fluorescence signal is less than the third fluorescence threshold but greater than the fourth fluorescence threshold, the lentiviral vector is considered to have partial payload fluorescence. In some embodiments, the first fluorescence threshold, the second fluorescence threshold, and the third fluorescence threshold are predefined. In some embodiments, the first fluorescence threshold, the second fluorescence threshold, and the third fluorescence threshold are calibrated. In some embodiments, the amount of payload required in each lentiviral vector can vary based on the amount of payload to be delivered and the disease to be treated. Thus, the third fluorescence threshold and optionally the fourth fluorescence threshold for different RNA payloads will also vary.

[0057] For each identified or detected lentiviral vector from the IM image, when all three fluorescence images have fluorescence signals at the same location (colocalization), the fluorescence signal is determined to correspond to a fully loaded lentiviral vector because these images contain fluorescence signals of envelope protein, capsid protein, and payload. When the first fluorescence image and the second fluorescence image have fluorescence signals at the same location, but the third fluorescence image does not have a fluorescence signal at the corresponding location, the fluorescence signal is determined to correspond to an empty lentiviral vector because the first fluorescence image and the second fluorescence image contain fluorescence signals of envelope protein and capsid protein, but do not contain a fluorescence signal of payload. When the first fluorescence image and the second fluorescence image have fluorescence signals at the same location, but the third fluorescence image has a fluorescence signal that is lower than the third threshold but greater than the fourth threshold, the fluorescence signal is determined to correspond to a partially loaded lentiviral vector because the first fluorescence image and the second fluorescence image contain fluorescence signals of envelope protein and capsid protein, but these images only contain a weak signal of payload. In some embodiments, other types of lentiviral vectors can be defined using their IM signals as well as the first fluorescence signal, the second fluorescence signal, and the third fluorescence signal.

[0058] In some embodiments, the characterization of the lentiviral vector includes: determining a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal; calculating the ratio between the number of lentiviral vectors having the first fluorescence signal, the second fluorescence signal, and the third fluorescence signal (fully loaded lentiviral vectors) and the number of lentiviral vectors having the first fluorescence signal (or the number of lentiviral vectors identified from the IM image, i.e., the total number of all lentiviral vectors). This ratio indicates the percentage of the number of fully loaded lentiviral particles to the total number of lentiviral particles.

[0059] In certain aspects, the present disclosure provides a method for optimizing lentiviral vector production by observing each layer of lentiviral morphology through a combination of process improvement steps and a novel analytical method using high-throughput fluorescence detection.

[0060] As discussed above, the present disclosure targets the lentiviral vector at three separate levels (envelope, capsid, and payload) to determine the composition of the vector particles. Through an envelope capture antibody, suitably via an anti-VSV-G capture antibody, the lentiviral sample is captured on a microarray chip. The bound sample is fixed, permeabilized, and stained with a mixture containing fluorescent antibodies (which target the envelope and capsid, such as the VSV-G envelope and the p24 capsid) and a permeable fluorescent nucleic acid stain that selectively targets the payload (such as an RNA payload). The stained sample is then scanned on a fluorescence imaging platform (e.g., an imaging platform), where the co-localization of all three fluorescence signals is monitored. The ratio of fully formed lentiviral vectors, empty lentiviral vectors, and incomplete particles is obtained from the fluorescence images. Based on this, the present invention further develops a protocol for optimizing process parameters, including combining certain components at specific ratios to increase productivity and plasmid design to identify parameters critical to the process. In addition, the present disclosure obtains a predictive assessment of lentiviral functionality, which helps to optimize the process to maximize yield. This data-driven protocol accelerates the process development speed.

[0061] In some embodiments, the optimization method comprises the following steps: predefining the ratios of the packaging plasmid, the envelope plasmid, the transfer plasmid, and the payload; for each ratio, transducing host cells using the ratio of the plasmids; culturing the transduced cells; obtaining a lentiviral vector population from the cell culture; determining the ratio of fully loaded lentiviral vectors or the total amount of fully loaded lentiviral vectors in the lentiviral vector population; selecting the ratio with the highest ratio or highest amount of fully loaded lentiviral vectors as the selected ratio; and using the selected ratio to produce lentiviral vectors. In some embodiments, the above optimization procedure may be repeated, for example, by defining a new set of ratios based on the selected ratio and obtaining a further selected ratio. After a predetermined number of repetitions or until there is no significant difference between the selected ratios in the last two repetitions, the final selected ratio is determined as the optimized ratio and can be used for the efficient production of lentiviral vectors.

[0062] In some embodiments, in addition to plasmid ratio optimization, the above optimization process can also be used to optimize different plasmid designs, optimize transduction conditions, optimize culture conditions, optimize ion exchange chromatography for purifying lentiviral vectors, or evaluate size exclusion steps for removing damaged lentiviral vectors.

[0063] In some embodiments, the above optimization process is suitable for high-throughput implementation, where a multi-well plate can be used to accommodate multiple microarrays, and each microarray is used for one experimental test. In some embodiments, a microarray can have multiple test points, and each test point is configured for one experimental test. In some embodiments, all or some of the optimization steps are automated to increase the efficiency of the process.

[0064] In some embodiments, the present disclosure provides a method for producing lentiviral vectors, wherein the production is carried out using the optimized conditions as described above.

[0065] Examples

[0066] In a first embodiment, provided herein is a method for characterizing a lentiviral vector, the method comprising: providing a sample comprising a population of lentiviral vectors, the population of lentiviral vectors comprising fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors; contacting a substrate with the sample such that the population of lentiviral vectors is captured on the substrate, wherein the substrate comprises an anti-envelope protein antibody; contacting the captured population of lentiviral vectors with a first fluorophore, a second fluorophore, and a third fluorophore, the first fluorophore comprising a first fluorescent label and a first binding molecule that binds to the envelope protein of the lentiviral vector, the second fluorophore comprising a second fluorescent label and a second binding molecule that binds to the capsid protein of the lentiviral vector, the third fluorophore comprising a third fluorescent label that binds to the payload of the lentiviral vector; irradiating the captured population of lentiviral vectors with light from an irradiation source so as to excite the first fluorophore, the second fluorophore, and the third fluorophore; detecting a first fluorescent light of a first fluorescent wavelength emitted from the first fluorophore, a second fluorescent light of a second fluorescent wavelength emitted from the second fluorophore, and a third fluorescent light of a third fluorescent wavelength emitted from the third fluorophore; and characterizing the lentiviral vector based on the detected first fluorescent light, second fluorescent light, and third fluorescent light, wherein the first fluorescent wavelength, the second fluorescent wavelength, and the third fluorescent wavelength are different.

[0067] Example 2 includes the method according to Example 1, wherein the substrate comprises an anti-vesicular stomatitis virus G protein (anti-VSV-G) antibody.

[0068] Example 3 includes the method according to Example 1 or 2, wherein the first binding molecule comprises an anti-VSV-G antibody, an anti-fusion protein (anti-F) antibody, an anti-hemagglutinin (anti-HA) antibody, or an anti-hemagglutinin neuraminidase (anti-HN) antibody.

[0069] Example 4 includes the method according to any one of Examples 1 to 3, wherein the second binding molecule comprises an anti-p7 antibody, an anti-p24 antibody, or an anti-p27 antibody.

[0070] Example 5 includes the method according to any one of Examples 1 to 4, wherein the third fluorescent label specifically binds to a nucleic acid or a protein.

[0071] Example 6 includes the method according to Example 5, the third fluorescent label comprising an anti-integrase antibody, an anti-reverse transcriptase antibody, or an anti-protease antibody.

[0072] Example 7 includes the method according to any one of Examples 1 to 6, wherein detection of the first fluorescent light ray and the second fluorescent light ray characterizes the empty lentiviral vector, and detection of the first fluorescent light ray, the second fluorescent light ray, and the third fluorescent light ray characterizes the fully loaded lentiviral vector.

[0073] Example 8 includes the method according to any one of Examples 1 to 7, wherein the substrate is a microarray chip.

[0074] Example 9 includes the method according to any one of Examples 1 to 8, wherein the fully loaded lentiviral vector contains ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0075] Example 10 includes the method according to any one of Examples 1 to 9, wherein each of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range from about 250 nm to about 700 nm.

[0076] Example 11 includes the method according to Example 10, wherein one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range from about 460 nm to about 510 nm.

[0077] Example 12 includes the method according to Example 10 or 11, wherein one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range from about 520 nm to about 570 nm.

[0078] Example 13 includes the method according to any one of Examples 10 to 12, wherein one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range from about 640 nm to about 680 nm.

[0079] Example 14 includes the method according to any one of Examples 1 to 13, wherein characterizing the lentiviral vector includes: determining a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal according to the intensities of the first fluorescent light ray, the second fluorescent light ray, and the third fluorescent light ray; and calculating a ratio between the area having the first signal, the second signal, and the third signal and the area having the first signal, wherein the ratio indicates the percentage of the fully loaded lentiviral vector in the sample relative to the total amount of lentiviral vectors in the sample.

[0080] Example 15 includes the method according to any one of Examples 1 to 14, wherein the captured population of lentiviral vectors is simultaneously contacted with the first fluorescent agent, the second fluorescent agent, and the third fluorescent agent.

[0081] Example 16 includes the method according to any one of Examples 1 to 15, wherein contacting the captured lentiviral vector population with the first fluorophore, the second fluorophore, and the third fluorophore comprises: contacting the captured lentiviral vector population with the third fluorophore, and then contacting the captured lentiviral vector population with the first fluorophore and the second fluorophore.

[0082] Example 17 includes the method according to any one of Examples 1 to 16, wherein contacting the captured lentiviral vector population comprises incubating the sample with the first fluorophore, second fluorophore, and third fluorophore for at least 15 minutes.

[0083] Example 18 includes the method according to any one of Examples 1 to 17, wherein the third fluorophore comprises a permeable fluorescent nucleic acid stain.

[0084] Example 19 includes the method according to any one of Examples 1 to 18, wherein the method further comprises permeabilizing the lentiviral vector population prior to contacting with the first fluorophore, the second fluorophore, and the third fluorophore.

[0085] Example

[0086] Example 1: Characterization of Lentiviral Vectors by Three-Layer Fluorescence Detection

[0087] A fluorescence device is provided, the fluorescence device comprising: three laser coherent light sources configured to generate light at, for example, approximately 552 nm, approximately 637 nm, and approximately 500 nm, respectively; one or more fluorescence light detectors configured to detect fluorescence light at, for example, approximately 590 nm, approximately 685 nm, and approximately 525 nm, respectively; an interference microscope for collecting IM images; and a computing device configured to process the images collected by the fluorescence light detector and the interference microscope. A microarray is provided having 24 (4×6) wells. Each well has a surface to which an anti-VSV-G antibody is immobilized. These wells are used to accommodate replicate tests of samples and / or to accommodate lentiviral populations generated using different parameters.

[0088] Prepare an RNA payload and the first fluorophore, the second fluorophore, and the third fluorophore. The first fluorophore comprises an anti-VSV-G antibody conjugated to a first fluorescent label. The first fluorescent label is a NovaFluor Yellow 590 dye having an excitation maximum of approximately 552 nm and an emission maximum of approximately 590 nm. The second fluorophore comprises an anti-p24 antibody conjugated to a second fluorescent label. The second fluorescent label is a NovaFluor Red 685 dye having an excitation maximum of approximately 637 nm and an emission maximum of approximately 685 nm. The third fluorescent label is Quant-iTTM RiboGree, which has an excitation maximum of approximately 500 nm and an emission maximum of approximately 525 nm.

[0089] Figure 3 Plasmid designs for balanced expression of packaging, envelope, and transgene for improved lentiviral vector packaging are shown. As Figure 3 shown, the constructed plasmids include two packaging plasmids, one envelope plasmid, and one transfer plasmid. The packaging plasmids contain the GAG-POL gene and the REV gene, the envelope plasmid contains the VSV-G gene, and the transfer plasmid contains the green fluorescent protein (GFP) gene as an exemplary GOI.

[0090] HEK-293 cells are provided. The HEK-293 cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C, 5% CO2, and 60% humidity until the desired confluence is reached. The cultured HEK-293 cells are transiently transfected with the packaging plasmid, the envelope plasmid, the transfer plasmid, and the RNA payload. For pVSV-G:pREV:pGAG-POL:pGOI / GFP, the molar ratio between the three types of plasmids and the payload plasmid is 1:1:2:3. The transfected HEK-293 are further cultured, and a sample with a lentiviral population is obtained from the transfected HEK-293 cells. The number of particles in the sample is estimated, for example, by interference microscopy or other suitable means. If necessary, the sample is diluted such that the concentration of the diluted sample contains about 10 6 to about 10 8 lentiviral particles.

[0091] The microarray is placed in the wells of a plate, and 25 μl of the diluted sample is placed on the microarray. The microarray loaded with the diluted sample is incubated at 20 °C for one hour. The diluted sample includes a lentiviral vector population. The lentiviral vector population can include fully loaded lentiviral vectors, partially loaded lentiviral vectors, empty lentiviral vectors, and small aggregates. The fully loaded lentiviral vectors have an envelope formed by the VSV-G protein, a capsid located inside the envelope and formed by the capsid protein, and an RNA payload packaged inside the capsid. The partially loaded lentiviral vectors have an envelope and a capsid, but the capsid contains only fragments of the RNA payload or an insufficient amount of the RNA payload and / or partial vector or host cell DNA / RNA. The empty lentiviral vectors contain an envelope and a capsid but substantially no RNA payload. The small aggregates can be formed only by the envelope protein or optionally only by the capsid protein. Since the microarray surface is immobilized with anti-VSV-G antibodies, the lentiviral particles with VSV-G protein are immobilized on the surface by binding to the anti-VSV-G antibodies on the microarray surface.

[0092] Remove the sample solution and leave the microchip to dry for about 5 minutes. Add 80 μl of each of the three fluorescent agents to immerse the microarray in the wells. Incubate the plate at 37 °C with shaking at 50 revolutions per minute (rpm) for 30 minutes and protect from light.

[0093] Scan the microarray with an interference microscope, and the scanned interference microscopy (IM) image shows the number of particles in the scanned area. Particles with a size less than 10 nm (diameter) are discarded; particles with a size of about 10 nm to about 50 nm are labeled as aggregates; particles with a size of about 50 nm to about 200 nm are labeled as lentiviral vectors.

[0094] Excitation fluorescence light is directed onto the microarray surface at wavelengths of 552 nm, 637 nm, and 500 nm, respectively. The emitted fluorescence light from the microchip surface is collected by one or more detectors of the fluorescence device at wavelengths of about 590 nm, about 685 nm, and about 525 nm, respectively, to obtain a first fluorescence image, a second fluorescence image, and a third fluorescence image.

[0095] In some embodiments, the fluorescence device does not include an interference microscope, and the lentiviral vector is directly identified from the first fluorescence image. In some embodiments, the lentiviral vector is identified by an image target detection algorithm. For example, if the fluorescence intensity of each pixel in the first fluorescence image is greater than a threshold, it is evaluated as a fluorescent pixel. Adjacent fluorescent pixels are combined to form a fluorescent spot, and if the longest diameter of the spot is greater than 50 nm, the spot represents a lentiviral vector. In some embodiments, the lentiviral vector can also be detected from each of the first fluorescence image, the second fluorescence image, and the third fluorescence image by an image target detection algorithm.

[0096] In some embodiments, the lentiviral vector is identified from the IM image and also by image target detection from all three fluorescence images. The identified or detected lentiviral vectors may overlap, and there may also be lentiviral vectors that are identified only from the IM image or only from one of the three fluorescence images.

[0097] After obtaining the IM image, the fluorescence images, and the identified lentiviral vectors, the computing device overlays the IM image on the first fluorescence image, the second fluorescence image, and the third fluorescence image. For each identified lentiviral vector in the IM image, the computing device calculates a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal from the first fluorescence image, the second fluorescence image, and the third fluorescence image corresponding to the identified lentiviral vector, and compares the first fluorescence signal, the second fluorescence signal, and the third fluorescence signal with a first fluorescence threshold, a second fluorescence threshold, and a third fluorescence threshold. When the first fluorescence signal (intensity) is greater than the first fluorescence threshold, the identified lentiviral vector is considered to have envelope fluorescence. When the second fluorescence signal (intensity) is greater than the second fluorescence threshold, the identified lentiviral vector is considered to have capsid fluorescence. When the third fluorescence signal (intensity) is greater than the third fluorescence threshold, the identified lentiviral vector is considered to have payload fluorescence; and when the third fluorescence signal is less than the third fluorescence threshold but greater than a fourth fluorescence threshold, the lentiviral vector is considered to have partial payload fluorescence. In some embodiments, the first fluorescence threshold, the second fluorescence threshold, and the third fluorescence threshold are predefined. In some embodiments, the first fluorescence threshold, the second fluorescence threshold, and the third fluorescence threshold are calibrated. In some embodiments, the amount of payload required in each lentiviral vector can vary based on the amount of payload being required to be delivered and the disease to be treated. Accordingly, the third fluorescence threshold for different RNA payloads will also vary.

[0098] The computing device then determines a fully loaded lentiviral vector if it has envelope fluorescence, capsid fluorescence, and payload fluorescence; determines an empty lentiviral vector if it has envelope fluorescence and capsid fluorescence but no payload fluorescence; and determines an impurity lentiviral vector if it has other IM / fluorescence patterns. Impurity lentiviral vectors can include partially loaded lentiviral vectors having envelope fluorescence, capsid fluorescence, and partial payload fluorescence, and aggregate lentiviral vectors having only envelope fluorescence.

[0099] In some embodiments, the computing device further counts the total number of lentiviral vectors and optionally aggregates from the IM image, counts the total number of fully loaded lentiviral vectors, and calculates the percentage of the total number of fully loaded lentiviral vectors to the total number of lentiviral vectors and / or aggregates. This percentage strongly indicates whether the host cells transiently transduced with the plasmid are suitable for lentiviral vector production.

[0100] In some embodiments, the ratios between the fluorescence intensities of the corresponding spots in the first image, the second image, and the third image are used to characterize the lentiviral vectors.

[0101] Example 2: Optimization of Lentiviral Vector Yield Based on Three-Layer Fluorescence Detection

[0102] As described in Example 1, a fluorescent device and a microarray are provided. As described in Example 1, an RNA payload, as well as a first fluorophore, a second fluorophore, and a third fluorophore are prepared. As described in Example 1, a packaging plasmid, an envelope plasmid, and a transfer plasmid are constructed. As described in Example 1, HEK-293 cells are provided. The difference lies in that the first fluorescent label (linked to the anti-VSV-G antibody), the second fluorescent label (linked to the anti-p24 antibody), and the third fluorescent label (targeting the RNA payload) are green fluorescence, red fluorescence, and blue fluorescence, respectively. Further, the parameters of Example 1, such as upstream optimization (which includes plasmid design and plasmid ratio) and downstream optimization (such as an ion exchange (IEX) polishing step for complete lentiviral vector enrichment and a size exclusion step for damaged lentiviral vector particles) can be varied in order to determine the optimal parameters for generating fully loaded lentiviral vectors.

[0103] In this Example 2, three sets of molar ratios between the packaging plasmid, the envelope plasmid, the transfer plasmid, and the RNA payload are predefined. In the first set, the molar ratio between the envelope plasmid (VSV-G), the packaging plasmid (REV), the packaging plasmid (GAG-POL), and the transfer plasmid expressing the RNA payload is 1:1:2:3; in the second set, the molar ratio is 1:1:2:1; in the third set, the molar ratio is 1:1:2:0.1.

[0104] As shown in Example 1, for each ratio, HEK-293 cells are cultured and transfected with the plasmids and the RNA payload at the ratios described above; the transfected cells are further cultured; the lentiviral vector population is recovered from the further cultured cells; the lentiviral vector population is incubated with the microchip; the incubated microchip is further incubated with the three fluorophores; the lentiviral vectors are scanned by interference microscopy; a laser light source with three excitation wavelengths is used to excite the further incubated microchip; the fluorescent light rays with three different emission wavelengths are collected and processed in order to determine whether the lentiviral vectors are fully loaded, partially loaded, or empty. Then the ratio of fully loaded lentiviral vectors in the total lentiviral vectors, or the total number of fully loaded lentiviral vectors, is obtained.

[0105] Since the microarray can be designed with multiple wells, three different sets of conditions can be characterized simultaneously. For example, a 4×6 well plate is provided, which has 4 rows and 6 columns. Each well is loaded with a microarray. 25 μl of a sample corresponding to the plasmids at the first ratio, which has two different dilutions such that the expected lentiviral vector concentration is about 10 6 to about 10 8 / ml, and each dilution is provided with three replicates. Similarly, six microarrays in the second row of the plate are added with samples corresponding to the plasmid of the second ratio, and six microarrays in the third row of the plate are added with samples corresponding to the plasmid of the third ratio. Some of the microarrays in the fourth row are designed as positive and negative controls, where the positive control uses a confirmed amount of lentiviral vector as the sample, and the negative control uses the culture medium (without cultured cells) as the sample.

[0106] In some embodiments, a microchip having a plurality of wells can be configured to be processed using a robotic device. In this way, optimization can be carried out in a high-throughput manner.

[0107] Figure 5 The results of Example 2 are shown, where the complete lentiviral vector has all the fluorescence signals of the envelope, capsid, payload, and IM signal, while the empty lentiviral vector does not include the fluorescence signal of the payload RNA. As Figure 5 shown, the 1:1:2:3 molar ratio provided 26.9% of the complete lentiviral vector, which is more favorable than the 1:1:2:1 molar ratio that provided 20.8% of the complete lentiviral vector, and more favorable than the 1:1:2:0.1 molar ratio that provided 6.2% of the complete lentiviral vector. Thus, the complete characterization of the lentiviral vector indicates that, among other things, a reduction in nucleic acid content during the production phase reduces the fraction of generated lentiviral particles.

[0108] Example 3: Optimization of lentiviral vector production based on three-layer fluorescence detection

[0109] Example 3 is similar to Example 2. Example 3 tested 5 plasmid ratios instead of three ratios, and the GOI was set to GFP. As Figure 6 shown, the molar ratio 1:1:2:3 is the most promising, where the complete lentiviral vector production is 15.8%.

[0110] In summary, the present disclosure provides fluorescence detection of all key components of the lentiviral construct, including the payload that is important for complete functional lentiviral particles; the present disclosure also provides a protocol for process optimization, including combining certain components in specific ratios to optimize productivity; and the present disclosure provides an analytical model for optimizing the process based on changes in process inputs to maximize yield. Further, the disclosed optimization process can be carried out in a high-throughput manner and without sample pretreatment.

[0111] It should be understood that although certain embodiments have been shown and described herein, the claims are not limited to the specific forms or arrangements of the parts described and shown. Illustrative embodiments have been disclosed in this specification, and although specific terms have been employed, they are used in a general and descriptive sense only and not for purposes of limitation. Modifications and variations of the described embodiments are possible in light of the above teachings. Accordingly, it should be understood that the embodiments may be practiced in a manner different from that specifically described.

[0112] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for characterizing a lentiviral vector, the method comprising: Providing a sample comprising a population of lentiviral vectors, the population of lentiviral vectors comprising fully loaded lentiviral vectors, partially loaded lentiviral vectors, and / or empty lentiviral vectors; Contacting a substrate with the sample so as to capture the population of lentiviral vectors on the substrate, wherein the substrate comprises an anti-envelope protein antibody; Contacting the captured population of lentiviral vectors with a first fluorophore, a second fluorophore, and a third fluorophore, the first fluorophore comprising a first fluorescent label and a first binding molecule that binds to the envelope protein of the lentiviral vector, the second fluorophore comprising a second fluorescent label and a second binding molecule that binds to the capsid protein of the lentiviral vector, and the third fluorophore comprising a third fluorescent label that binds to the payload of the lentiviral vector; Irradiating the captured population of lentiviral vectors with light from an irradiation source so as to excite the first fluorophore, the second fluorophore, and the third fluorophore; Detecting a first fluorescent light of a first fluorescent wavelength emitted from the first fluorophore, a second fluorescent light of a second fluorescent wavelength emitted from the second fluorophore, and a third fluorescent light of a third fluorescent wavelength emitted from the third fluorophore; and Characterizing the lentiviral vector based on the detected first fluorescent light, second fluorescent light, and third fluorescent light, wherein the first fluorescent wavelength, the second fluorescent wavelength, and the third fluorescent wavelength are different.

2. The method according to claim 1, wherein the substrate comprises an anti-vesicular stomatitis virus G protein (anti-VSV-G) antibody.

3. The method according to claim 1 or claim 2, wherein the first binding molecule comprises an anti-VSV-G antibody, an anti-fusion protein (anti-F) antibody, an anti-hemagglutinin (anti-HA) antibody, or an anti-hemagglutinin neuraminidase (anti-HN) antibody.

4. The method according to any one of claims 1 to 3, wherein the second binding molecule comprises an anti-p7 antibody, an anti-p24 antibody, or an anti-p27 antibody.

5. The method according to any one of claims 1 to 4, wherein the third fluorescent label specifically binds to nucleic acid or protein.

6. The method according to claim 5, wherein the third fluorescent label comprises an anti-integrase antibody, an anti-reverse transcriptase antibody, or an anti-protease antibody.

7. The method according to any one of claims 1 to 6, wherein detection of the first fluorescent light and the second fluorescent light characterizes the empty lentiviral vector, and detection of the first fluorescent light, the second fluorescent light, and the third fluorescent light characterizes the fully loaded lentiviral vector.

8. The method according to any one of claims 1 to 7, wherein the substrate is a microarray chip.

9. The method according to any one of claims 1 to 8, wherein the fully loaded lentiviral vector comprises ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

10. The method according to any one of claims 1 to 9, wherein each of the first fluorescent wavelength, the second fluorescent wavelength, and the third fluorescent wavelength is in the range from about 250 nm to about 700 nm.

11. The method according to claim 10, wherein one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range of about 460 nm to about 510 nm.

12. The method according to claim 10 or claim 11, wherein one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range of about 520 nm to about 570 nm.

13. The method according to any one of claims 10 to 12, wherein one of the first fluorescence wavelength, the second fluorescence wavelength, and the third fluorescence wavelength is in the range of about 640 nm to about 680 nm.

14. The method according to any one of claims 1 to 13, wherein characterizing the lentiviral vector comprises: determining a first fluorescence signal, a second fluorescence signal, and a third fluorescence signal based on the intensities of the first fluorescence light, the second fluorescence light, and the third fluorescence light; and calculating a ratio between the area having the first signal, the second signal, and the third signal and the area having the first signal, wherein the ratio indicates the percentage of the fully loaded lentiviral vectors in the sample relative to the total amount of lentiviral vectors in the sample.

15. The method according to any one of claims 1 to 14, wherein the captured population of lentiviral vectors is contacted simultaneously with the first fluorophore, the second fluorophore, and the third fluorophore.

16. The method according to any one of claims 1 to 15, wherein contacting the captured lentiviral vector population with the first fluorophore, the second fluorophore, and the third fluorophore comprises: The captured population of lentiviral vectors is contacted with the third fluorophore, and then the captured population of lentiviral vectors is contacted with the first fluorophore and the second fluorophore.

17. The method according to any one of claims 1 to 16, wherein contacting the captured population of lentiviral vectors comprises incubating the sample with the first fluorophore, the second fluorophore, and the third fluorophore together for at least 15 minutes.

18. The method according to any one of claims 1 to 17, wherein the third fluorophore comprises a permeable fluorescent nucleic acid stain.

19. The method according to any one of claims 1 to 18, the method further comprising permeabilizing the population of lentiviral vectors before contacting with the first fluorophore, the second fluorophore, and the third fluorophore.

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